Isolation And Culture Of Human Embryonic Germ Cells And Their Biological Characteristics

Primordial germ cells (PGCs), which are the embryonic precursors of gametes or the progenitor cells which produce sperms and eggs at later stages of development. Embryonic germ (EG) cells are pluripotent cells derived from the primordial germ cells of gonads, gonadal ridges and mesenteries, and analogies of foetuses, with the ability to undergo both self-renewal and multiple differentiation. These cells can differentiate into derivatives ofall three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cell of the body. Human embryonic germ cells grow highly like mouse embryonic stem or EG cells in appropriate environment. Great progress have been achieved in the isolation and culture of mouse EG cells at present.But there are little information about human germ cells development and differentiation,specific markers during human embryo gestation.,specific factors that infuencing isolation and culture ,and the using of serum-free culture for human EG cells , specific differentiation of human EG cells. In present study, PGCs have already been isolated and cultured, In order to establish a human embryonic germ cell line and investigate its specific differntiation,and human cardiac α-actin promoter driving pEGFP-N1 was transfected into human EG cells so as to improve and purify cardiomyocytes derived from human EG cells. This thesis consists of five parts: 1. Tracing some markers during human embryo gestation; 2. Building of appropriate system for the establishment of human EG cell lines ; 3. Serum-free culture for human EG cells and their identification; 4. Specific differentiation of human EG cells; 5. Transfection of human EG cells with human cardiac α-actin promoter driving pEGFP-N1 . The conclusion: 1. Human embryo testis and ovary initially developed at 11.5 week and 20 week. The results demonstrate Ki67 expressed strongly in 10 week testis and 20 week ovary; Bcl-2 expressed strongly in 11.5 week testis , but low in ovary; human PGCs with large and prominent nuclei, usually express Oct4,AKP,SSEA1 before 12 week testis and ovary. Gradually, Oct4, AKP and SSEA1 expression decline till 32 week. Transmission electron microscope(TEM) show big PGCs with round or elliptic nuclei, high ratio of nuclei to cytoplasm, and some characteristic organelles of PGCs like ribosomes, endoplasmic reticulum and mitochondrion. With sex differentiation, mesenchymal cells and Sertoli cells appeared in embryoic testis and granulosa cells and oocytes in embryoic ovary. 2. Human EG cell lines can be clonally isolated from PGCs. Factors influencing the efficiency of isolation and culture of human PGCs, such as foetus age, serum, methods of isolation and propagation, extracellular matrix, cytokines , conditioned medium(CM) of BRL, RC, feeder cells and other supplements was investigated in the paper. The results demonstrate that foetus over 7–12 week are optimal for in vitro culture of human EG cells. The efficiency of hEGCs lines from male fetus is higher than from female.The basic medium consisted of DMEM, 0.1mmol/L non-essential amino acids, 0.1mmol/L β-mercaptoethanol, 2mmol/L L-glutamine and 1mmol/L sodium pyruvate. Supplementation with 15%KSR, 5 ng/mL human recombinant leukaemia inhibitory factor, 10 ng/mL basic fibroblast growth factor and 10μmol/L Forskolin clearly improve the efficiency of isolation and derivation of human EG cells. Murine embryonic fibroblasts(MEF) and STO cell line are better feeder cells than human embryonic fibroblasts, human fetal(HEF),bone marrow-derived MSCs(hMSCs) or bovine embryonic fibroblasts. Supplement fibronectin(FN),collagen Ⅳ,and 0.1% gleatin could enhance growth rate of AKP positive of hEGCs. 3. The human EG cells were cultured on MEF or STO feeder layer with serum-free culture medium and maintained undifferentiated state for 14 passages. Human EG cells maintained undifferentiated state better in knock-out serum replacement (KSR) than in FBS medium. The hEGCs clones were typically like mouse ES clones in morphology, tight, compact, multilayer of cells with large and prominent nuclei, showed normal and stable diploid karyotype and pluripotency to differentiate into cells of all three embryonic germ layers, express AKP, SSEAs and glycoprotein such as SSEA-1, SSEA-3, SSEA-4,TRA-1-60,TRA-1-81,and express Oct-4, some expressing PCNA ,C-kit,and with high telomerase activity.In contrast,mouse EGCs express AKP, SSEA1, Oct-4,exressing PCNA, C-kit,and with high telomerase activity,but not SSEA-3, SSEA-4, TRA-1-60,TRA-1-81. During human EG cells in vitro suspension culture, delaying passage or growing beyond confluence of fibroblast feeder layers, Human EG cells can form simple and cystic embryoid bodies (EBs) consists of various cell types including all three embryonic germ layers, such as neural cells, epithelia-like, fobroblast-like or polygon cells and rhthymically beating myocardium-like cells ,even sperm and oocyte-like,and so on. The cultured cells have been continuausly passaged. In conclusion, we obtain pluripotent human EG cell lines derived from PGCs. 4. Human EG cells were induced and differentiated into cardiomyocytes, neural cells and insulin-secreting cells.The results demonstrate that 0.75%DMSO,10-7 mol/L RA or 10 μmol/L 5-azacytidine and 40% cardiomyocytes conditioned medium can increase the positive rate of human cardiac α-actin, PAS positive cardiomyocyte-like cells. Amnion and 10-6mol/L RA orβ-mercaptoethanol (1 mmol/L ,2.5 mmol/L ,5 mmol/L) can enhance the number of neural cells derived from EBs of human EG cells. 10 ng/mL HGF+ 10 umol/L nicotinamide can improve the amount of insulin-secreting cells derived from EBs of human EG cells. Results suggest that human EG cells could be a good source for celltherapy for heart infraction, neural injured diseases and diabetes. 5. In order to enhance the differentiation rate of human EG cells into cardiomyocytes, we construct expression vector, namedα-actin-pEGFP-N1 which consists of neor gene and EGFP gene as positive selection markers. Then transfer it into human EG cells by Lipofectamine2000. Fluorescent observation and immunocytochemistry assay demonstrated that human cardiac α-actin promoter was expressed and had strong regulative capacity in human EG cells. The transfected cells show high levels ofα-actin and GFP expression,easily induced differentiation ability into cardiomyocytes-like cells compared with wild human EG cells , such as more PAS positive cells in transfected cells than human EG cells , grow up for 3 passages. The results suggest that human cardiac α-actin promoter may be useful in the study of human EG cells specific differentiation into cardiomyocytes. A human EG cell line tranfected wih cardiac α-actin driving pEGFP-N1 was established. This will provide the means to the study on cardiogenesis, differentiation, especially study on manipulating these cells for the purpose of basic and applied research.